Cancer cells possess two properties which place an extreme burden on the DNA replication and repair machinery: they divide rapidly and are often polyploid. These characteristics may require novel strategies for DNA replication that are not utilized during normal cell division. As an example, the expression of DNA polymerase theta, an error-prone translesion polymerase, is frequently upregulated in cancer cells and this overexpression correlates with excessive chromosomal damage and a negative clinical outcome. We have recently found that Drosophila lacking polymerase theta have abnormal phenotypes and display genome instability in tissues that are characterized by rapid S phases and/or polyploidy, including early stage embryos, follicle cells in the female germline, and histoblasts. In addition, we have identified roles for translesion polymerases in homologous recombination repair These preliminary data establish Drosophila as an excellent system in which to investigate tissue- and cellspecific functions of translesion polymerases and provide an opportunity to test the hypothesis that translesion polymerases play important roles in cells that experience endogenous replication stress. We will utilize a novel lacZ reporter system to assess the frequency and types of mutations that arise during replication and homologous recombination repair when polymerase theta and other translesion polymerases are mutated or overexpressed. In addition, we will collaborate with the Lovett and Freudenreich labs to test whether trinucleotide repeats and sequences that form quasi-palindromes experience heightened instability when located near double-strand breaks or under conditions of endogenous replication stress. Together, these studies will significantly advance our long-term goal to understand how the use and misuse of translesion polymerases contributes to genome instability in cancer cells.